PSMAe/p特异性驱动shRNA靶向干扰前列腺癌LNCap细胞的研究

摘要

目的:探讨前列腺特异性膜抗原增强子/启动子(PSMAe/p)驱动短发夹RNA(shRNA)靶向干扰前列腺癌细胞的特异性.方法:运用RNA干扰技术,以转铁蛋白-聚乙二醇-聚乙烯亚胺(Tf-PEG-PEI)作为基因转移载体,以前列腺癌LNCap细胞为研究对象,并以前列腺癌PC-3细胞、膀胱癌T24细胞及人胚肾HEK293细胞做为对照,将外源增强绿色荧光蛋白(EGFP)基因及以PSMAe/p为启动序列靶向EGFP的干扰质粒转入培养细胞,以实时定量PCR(qRT-PCR)及Westernblot分别检测EGFP基因及蛋白在4种细胞各组中的表达情况,研究PSMAe/p驱动shRNA靶向干扰前列腺癌细胞的特异性.结果:在前列腺癌LNCap细胞组中,与单独转入EGFP基因(pEGFP-C1)的细胞组(A组)及转入EGFP基因(pEGFP-C1)+空载体质粒组(pPSMAe/p-UPRT)(C组)相比,转入EGFP基因(pEGFP-C1)及干扰质粒[pPSMAe/p-shEGFP-poly(A)]组(B组),EGFP基因表达及蛋白表达水平下调,分别与对照组(A组、C组)相比差异具有统计学差异(P<0.05)而转入EGFP基因(pEGFP-C1)的细胞组(A组)和转入EGFP基因(pEGFP-C1)+空载体质粒组pPSMAe/p-UPRT)(C组)间比较差异,无统计学意义(P>0.05);而在前列腺癌PC-3细胞、膀胱癌T24细胞及人胚肾HEK293细胞中EGFP基因及蛋白表达水平在实验组与对照组、对照组间比较差异无统计学意义(P>0.05).结论:PSMAe/p驱动shRNA具有细胞特异性,其可驱动特异基因片段在前列腺癌LNCap细胞中表达,从而实现基因治疗的细胞特异性,可作为前列腺癌基因治疗的靶向策略之一.%Objective: To investigate the cellular specificity of short-hairpin RNA ( shRNA ) transcription driven by the prostate-specific membrane antigen enhancer/promoter ( PSMAe/p ) in gene therapy for prostate cancer. Methods: The exogenous enhanced green fluorescent protein ( EGFP ) gene and shRNA interference fragments that target EGFP driven by PSMAe/p were cotrans-fected into prostate cancer LNCap cells using RNA interference technology. Transferrin-polyethylene glycol-polyethyleneimine ( Tf-PEG-PEI) was used as the gene transfer vector, and prostate cancer PC-3 cells, bladder cancer T-24 cells, human embryonic kidney HEK293 cells were used as the controls. Quantitative real time- polymerase chain reaction (PCR) and western blot analysis were used to detect EGFP gene mRNA and protein expression. Results: In the LNCap cell groups, compared with group A (the cells transfected with pEGFP-Cl ) and group C ( the cells cotransfected with pEGFP-Cl and the empty pPSMAe/p-UPRT plasmid ), EGFP gene mRNA and protein expression in group B significantly decreased after the LNCap cells were cotransfected with pEGFP-Cl and the interference plasmid pPSMAe/p-shEGFP-poly( A ) ( P < 0.05 ). However, no significant differences were observed between group A and group C ( P> 0.05 ). In the PC-3, T24, and HEK293 cell lines, no significant differences in EGFP gene mRNA and protein expression were observed among the groups. Conclusion: The short-hairpin RNA transcription driven by PSMAe/p in prostate cancer LNCap cells has cellular specificity, which can be used as a strategy in prostate cancer gene therapy.